The Triggering Receptors Expressed on Myeloid Cells (TREM) are expressed on a variety of innate immune cells including monocytes, macrophages, dendritic cells (DC), neutrophils, and osteoclasts. These receptors deliver signals to their host cells via association with the signaling chain, DAP12. DAP12 signaling is dependent on the presence of an immunoreceptor tyrosine-based activation motif (ITAM) within its cytoplasmic tail. Upon stimulation of a DAP12-coupled receptor, DAP12 is phosphorylated and recruits proteins critical to the propagation of downstream signals. Recent work has demonstrated that members of the TREM family, via DAP12, can deliver either activation or inhibitory signals to monocytes and macrophages. However, the biochemical nature of DAP12 signaling within myeloid cells is largely uncharacterized as is the overall immunological role of the TREM gene cluster. Thus, we have taken a bipartite approach to understand the immunobiology of the TREM cluster. First of all we have been characterizing a novel member of the gene cluster we discovered several years ago, TREM-Like transcript 1 (TLT-1). This TREM-like gene encodes a receptor specifically expressed in megakaryocytes and peripheral blood platelets. Activation of the platelets results in a translocation of TLT-1 from the alpha granules of platelets to the cell surface. We have characterized TLT-1, and recently produced mice with a specific mutation in TLT-1. These TLT-1 null mice exhibit decreased platelet aggregation and as a consequence have more pronounced hemorrhage associated with the Shwartzman model of hemorrhagic vasculitis. In addition, we have identified putative ligands for TLT-1 and are continuing our characterization of TLT-1 and TLT-1 null mice in collaboration with Dr. Valance Washington. Our second approach to understanding the role of TREM in regulation of innate immunity and cancer is dissection of the DAP12 signaling pathway in myeloid cells. Our studies have identified a shifting, developmentally regulated signaling cassette in macrophages and monocytes. Monocytes express two key intracellular adaptor proteins, the Linker for Activation of T cells (LAT) and the Linker for Activation of B cells (LAB, also known as the Non-T cell Adaptor, NTAL). We find that during maturation of DC or macrophages from monocytes in vitro, the levels of LAT fall whereas the levels of LAB increase. The result is that the signaling pathway utilized by DAP12 changes. Accordingly we have found that LAB is readily phosphorylated in response to DAP12 signaling. Moreover, analysis of LAB null mice, and macrophages derived from them, demonstrated hyperactive ITAM signaling and increased activation of the MAP kinase cascade. We have demonstrated that the ability of LAB to suppress ITAM signaling in macrophages likely derives from its ability to recruit the E3 ubiquitin ligase cCbl to the TREM receptor cluster. The lack of cCbl recruitment results in increased proximal ITAM signaling resulting in a developmental shift in the macrophages to a more immunosuppressive state characterized by lower production of IL-12/23 and higher production of IL-10 upon stimulation with LPS. Lastly, when tested in the Shwartzman reaction, LAB-/- have less inflammation than wild type mice suggesting the shift in macrophage polarity is physiologically relevant. In addition to our signaling studies in the myeloid compartment of the innate immune system we study the signaling of the Ly49 and KIR of natural killer cells, the lymphoid component of the innate immune system. The Ly49 and KIR families are comprised of both inhibitory and activating receptors; the latter interacting and signaling through DAP12. Thus we have been dissecting the ITAM signaling of NK cells with emphasis on on DAP12 signaling. We have discovered the parallel use of LAT and LAB in NK cells. Through the study mice lacking LAT, LAB, or both adaptors, we have demonstrated that the ITAM associated receptor NK1.1 can utilized either adaptor to elicit Interferon gamma (IFNgamma) production. Mast cells can also send some signals directly via LAB but the mechanism is unknown so we are dissecting the protein complexes associated with LAB in LAT null mice to further understand the nature of a LAB propagated signal. Toward this end we have developed a novel method for the delivery of signaling proteins or shRNAs to NK cells in vivo